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. 1996 Jul;70(7):4338–4344. doi: 10.1128/jvi.70.7.4338-4344.1996

The leucine domain of the visna virus Tat protein mediates targeting to an AP-1 site in the viral long terminal repeat.

L M Carruth 1, B A Morse 1, J E Clements 1
PMCID: PMC190366  PMID: 8676456

Abstract

The visna virus Tat protein is a strong transcriptional activator and is necessary for efficient viral replication. The Tat protein regulates transcription through an AP-1 site proximal to the TATA box within the viral long terminal repeat (LTR). Previous studies from our laboratory using Tat-Gal4 chimeric proteins showed that Tat has a potent acidic activation domain. Furthermore, a region adjacent to the Tat activation domain contains a highly conserved leucine-rich domain which, in the context of the full-length protein, suppressed the activity of the activation domain. To further elucidate the role of this region, four leucine residues within this region of Tat were mutated. In transient-transfection assays using visna virus LTR-CAT as a reporter construct, the activity of this leucine mutant was dramatically reduced. Additionally, domain-swapping experiments using the N-terminal activation domain of VP16 showed that the leucine-rich domain of Tat confers AP-1 responsiveness to the chimeric VP16-Tat protein. A chimeric VP16-Tat construct containing the leucine mutations showed no increased AP-1 responsiveness in comparison with that of the VP16 activation domain alone. Furthermore, in competition experiments, a Gal4-Tat protein containing only the leucine region of Tat (amino acids 34 to 62) was able to inhibit by competition the activity of full-length Tat. These studies strongly suggest that this leucine-rich domain is responsible for targeting the Tat protein to AP-1 sites in the viral LTR. In addition, examination of the amino acid sequence of this region of Tat revealed a highly helical secondary structure and a pattern of residues similar to that in the leucine zippers in the bZIP family of DNA-binding proteins. This has important implications for the interaction of Tat with cellular proteins, specifically Fos and Jun, that contain bZIP domains.

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Selected References

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  1. Abate C., Luk D., Curran T. Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains. Mol Cell Biol. 1991 Jul;11(7):3624–3632. doi: 10.1128/mcb.11.7.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
  3. Berkhout B., Silverman R. H., Jeang K. T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell. 1989 Oct 20;59(2):273–282. doi: 10.1016/0092-8674(89)90289-4. [DOI] [PubMed] [Google Scholar]
  4. Béraud C., Lombard-Platet G., Michal Y., Jalinot P. Binding of the HTLV-I Tax1 transactivator to the inducible 21 bp enhancer is mediated by the cellular factor HEB1. EMBO J. 1991 Dec;10(12):3795–3803. doi: 10.1002/j.1460-2075.1991.tb04949.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carruth L. M., Hardwick J. M., Morse B. A., Clements J. E. Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains. J Virol. 1994 Oct;68(10):6137–6146. doi: 10.1128/jvi.68.10.6137-6146.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choy B., Green M. R. Eukaryotic activators function during multiple steps of preinitiation complex assembly. Nature. 1993 Dec 9;366(6455):531–536. doi: 10.1038/366531a0. [DOI] [PubMed] [Google Scholar]
  7. Connor L. M., Oxman M. N., Brady J. N., Marriott S. J. Twenty-one base pair repeat elements influence the ability of a Gal4-Tax fusion protein to transactivate the HTLV-I long terminal repeat. Virology. 1993 Aug;195(2):569–577. doi: 10.1006/viro.1993.1408. [DOI] [PubMed] [Google Scholar]
  8. Davis J. L., Clements J. E. Characterization of a cDNA clone encoding the visna virus transactivating protein. Proc Natl Acad Sci U S A. 1989 Jan;86(2):414–418. doi: 10.1073/pnas.86.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Derse D., Carvalho M., Carroll R., Peterlin B. M. A minimal lentivirus Tat. J Virol. 1991 Dec;65(12):7012–7015. doi: 10.1128/jvi.65.12.7012-7015.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dingwall C., Ernberg I., Gait M. J., Green S. M., Heaphy S., Karn J., Lowe A. D., Singh M., Skinner M. A., Valerio R. Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6925–6929. doi: 10.1073/pnas.86.18.6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frankel A. D., Bredt D. S., Pabo C. O. Tat protein from human immunodeficiency virus forms a metal-linked dimer. Science. 1988 Apr 1;240(4848):70–73. doi: 10.1126/science.2832944. [DOI] [PubMed] [Google Scholar]
  12. Gabuzda D. H., Hess J. L., Small J. A., Clements J. E. Regulation of the visna virus long terminal repeat in macrophages involves cellular factors that bind sequences containing AP-1 sites. Mol Cell Biol. 1989 Jun;9(6):2728–2733. doi: 10.1128/mcb.9.6.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gdovin S. L., Clements J. E. Molecular mechanisms of visna virus Tat: identification of the targets for transcriptional activation and evidence for a post-transcriptional effect. Virology. 1992 Jun;188(2):438–450. doi: 10.1016/0042-6822(92)90497-d. [DOI] [PubMed] [Google Scholar]
  14. Gendelman H. E., Moench T. R., Narayan O., Griffin D. E., Clements J. E. A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues. J Virol Methods. 1985 Jun;11(2):93–103. doi: 10.1016/0166-0934(85)90033-3. [DOI] [PubMed] [Google Scholar]
  15. Gendelman H. E., Narayan O., Molineaux S., Clements J. E., Ghotbi Z. Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7086–7090. doi: 10.1073/pnas.82.20.7086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hardwick J. M., Tse L., Applegren N., Nicholas J., Veliuona M. A. The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16. J Virol. 1992 Sep;66(9):5500–5508. doi: 10.1128/jvi.66.9.5500-5508.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hess J. L., Small J. A., Clements J. E. Sequences in the visna virus long terminal repeat that control transcriptional activity and respond to viral trans-activation: involvement of AP-1 sites in basal activity and trans-activation. J Virol. 1989 Jul;63(7):3001–3015. doi: 10.1128/jvi.63.7.3001-3015.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hsu W., Kerppola T. K., Chen P. L., Curran T., Chen-Kiang S. Fos and Jun repress transcription activation by NF-IL6 through association at the basic zipper region. Mol Cell Biol. 1994 Jan;14(1):268–276. doi: 10.1128/mcb.14.1.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kamine J., Subramanian T., Chinnadurai G. Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8510–8514. doi: 10.1073/pnas.88.19.8510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kashanchi F., Piras G., Radonovich M. F., Duvall J. F., Fattaey A., Chiang C. M., Roeder R. G., Brady J. N. Direct interaction of human TFIID with the HIV-1 transactivator tat. Nature. 1994 Jan 20;367(6460):295–299. doi: 10.1038/367295a0. [DOI] [PubMed] [Google Scholar]
  21. Kerppola T. K., Luk D., Curran T. Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro. Mol Cell Biol. 1993 Jun;13(6):3782–3791. doi: 10.1128/mcb.13.6.3782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li Y., Mak G., Franza B. R., Jr In vitro study of functional involvement of Sp1, NF-kappa B/Rel, and AP1 in phorbol 12-myristate 13-acetate-mediated HIV-1 long terminal repeat activation. J Biol Chem. 1994 Dec 2;269(48):30616–30619. [PubMed] [Google Scholar]
  23. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  24. Luo Y., Peterlin B. M. Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR. J Virol. 1993 Jun;67(6):3441–3445. doi: 10.1128/jvi.67.6.3441-3445.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mohamed M. K., Tung L., Takimoto G. S., Horwitz K. B. The leucine zippers of c-fos and c-jun for progesterone receptor dimerization: A-dominance in the A/B heterodimer. J Steroid Biochem Mol Biol. 1994 Dec;51(5-6):241–250. doi: 10.1016/0960-0760(94)90036-1. [DOI] [PubMed] [Google Scholar]
  26. Narayan O., Cork L. C. Lentiviral diseases of sheep and goats: chronic pneumonia leukoencephalomyelitis and arthritis. Rev Infect Dis. 1985 Jan-Feb;7(1):89–98. doi: 10.1093/clinids/7.1.89. [DOI] [PubMed] [Google Scholar]
  27. Neuveut C., Vigne R., Clements J. E., Sire J. The visna transcriptional activator Tat: effects on the viral LTR and on cellular genes. Virology. 1993 Nov;197(1):236–244. doi: 10.1006/viro.1993.1584. [DOI] [PubMed] [Google Scholar]
  28. Ptashne M., Gann A. A. Activators and targets. Nature. 1990 Jul 26;346(6282):329–331. doi: 10.1038/346329a0. [DOI] [PubMed] [Google Scholar]
  29. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  30. Shih D. S., Carruth L. M., Anderson M., Clements J. E. Involvement of FOS and JUN in the activation of visna virus gene expression in macrophages through an AP-1 site in the viral LTR. Virology. 1992 Sep;190(1):84–91. doi: 10.1016/0042-6822(92)91194-y. [DOI] [PubMed] [Google Scholar]
  31. Small J. A., Bieberich C., Ghotbi Z., Hess J., Scangos G. A., Clements J. E. The visna virus long terminal repeat directs expression of a reporter gene in activated macrophages, lymphocytes, and the central nervous systems of transgenic mice. J Virol. 1989 May;63(5):1891–1896. doi: 10.1128/jvi.63.5.1891-1896.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sollerbrant K., Akusjärvi G., Linder S., Svensson C. The DNA binding domains of the yeast Gal4 and human c-Jun transcription factors interact through the zinc-finger and bZIP motifs. Nucleic Acids Res. 1995 Feb 25;23(4):588–594. doi: 10.1093/nar/23.4.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Southgate C. D., Green M. R. The HIV-1 Tat protein activates transcription from an upstream DNA-binding site: implications for Tat function. Genes Dev. 1991 Dec;5(12B):2496–2507. doi: 10.1101/gad.5.12b.2496. [DOI] [PubMed] [Google Scholar]
  34. Stringer K. F., Ingles C. J., Greenblatt J. Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature. 1990 Jun 28;345(6278):783–786. doi: 10.1038/345783a0. [DOI] [PubMed] [Google Scholar]
  35. Suckow M., Schwamborn K., Kisters-Woike B., von Wilcken-Bergmann B., Müller-Hill B. Replacement of invariant bZip residues within the basic region of the yeast transcriptional activator GCN4 can change its DNA binding specificity. Nucleic Acids Res. 1994 Oct 25;22(21):4395–4404. doi: 10.1093/nar/22.21.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tanimura A., Teshima H., Fujisawa J., Yoshida M. A new regulatory element that augments the Tax-dependent enhancer of human T-cell leukemia virus type 1 and cloning of cDNAs encoding its binding proteins. J Virol. 1993 Sep;67(9):5375–5382. doi: 10.1128/jvi.67.9.5375-5382.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tiley L. S., Brown P. H., Le S. Y., Maizel J. V., Clements J. E., Cullen B. R. Visna virus encodes a post-transcriptional regulator of viral structural gene expression. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7497–7501. doi: 10.1073/pnas.87.19.7497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vinson C. R., Hai T., Boyd S. M. Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design. Genes Dev. 1993 Jun;7(6):1047–1058. doi: 10.1101/gad.7.6.1047. [DOI] [PubMed] [Google Scholar]
  39. Walker S., Hayes S., O'Hare P. Site-specific conformational alteration of the Oct-1 POU domain-DNA complex as the basis for differential recognition by Vmw65 (VP16). Cell. 1994 Dec 2;79(5):841–852. doi: 10.1016/0092-8674(94)90073-6. [DOI] [PubMed] [Google Scholar]
  40. Xanthoudakis S., Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J. 1992 Feb;11(2):653–665. doi: 10.1002/j.1460-2075.1992.tb05097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xanthoudakis S., Miao G. G., Curran T. The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):23–27. doi: 10.1073/pnas.91.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]

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